Oviductal and uterine influence on the development of Day-2 equine embryos in vivo and in vitro

The objective of this experiment was to contrast the influence of the oviductal and uterine environments on development of Day-2 embryos. Embryos were transferred to oviducts or uteri of synchronous recipient mares, or were incubated in oviductal co-culture, in uterine co-culture or in defined culture medium. Significantly more (P < 0.02) embryos transferred to the oviduct versus the uterus survived until Day 11 after ovulation (5 7 vs 0 7 , respectively). Significantly more (P < 0.001) embryos developed to expanded and hatched blastocysts in uterine co-culture than in culture medium (6 7 vs 0 7 , respectively). The rate of embryo development to expanded blastocysts was not significantly different (P > 0.1) in oviductal co-culture versus uterine co-culture (3 7 vs 6 7 , respectively), or in oviductal co-culture versus culture in medium (3 7 vs 0 7 , respectively). Three of 7 and 6 of 7 embryos developed to hatched blastocysts greater than 2000 mum in diameter during oviductal and uterine co-culture, respectively, while 0 of 7 embryos cultured in medium expanded to greater than 500 mum in diameter. Proportions of embryos that developed for at least 9 days.     

Effect of one-step sucrose dilution of glycerol on in-vitro development of frozen-thawed mouse embyros

Several glycerol (GLY) dilution treatments were compared using frozen-thawed early blastocysts from Swiss Webster mice. These treatments consisted of 0.00 (0.00S n = 68), 0.25 (0.25S n = 67), 0.50 (0.50S n = 76), 0.75 (0.75S n = 66), 1.00 (1.00S n = 59), and 1.25 (1.25S n = 60) M of sucrose to remove GLY from embryos in one step. Then the one step procedure was compared with a three-step GLY dilution treatment (C n = 66). Embryos were exposed to 1.5 M of GLY in three-steps, frozen according to a standard freezing technique and stored at -196 degrees C. Embryos were thawed in a 37 degrees C water bath, pooled, and those graded good or excellent were randomly assigned to the experimental groups. The blastocysts were cultured in Whitten's medium microdrops under paraffin oil in a water saturated 5% CO(2) air atmosphere at 37 degrees C. The proportion (%) of embryos developing to expanded blastocysts was lowest (P < 0.05) in treatment 0.00S (63.1 +/- 4.0). The 0.25S (72.0 +/- 4.3) and 0.50S (75.0 +/- 3.1) 0.75S (79.0 +/- 4.4) treatments yielded a similar percentage of expanded blastocysts. The 1.00S treatment (87.0 +/- 4.0) was similar to 0.75S and 1.25S (98.3 +/- 4.0) treatments. The C treatment was superior (P < 0.05) to dilutions done with < 0.75 M sucrose, similar to 0.75S and 1.00S, and inferior (P < 0.05) to 1.25S. This later treatment yielded the highest percentage of expanded blastocysts. The percentage of embryos that hatched in treatments 0.00S, 0.25S, 0.50S, 0.75S and C was lower (P < 0.05) compared to 1.00S and 1.25S. The percentage of embryos and hatched blastocysts increased linearly (P < 0.01) from 0.0 to 1.25 M sucrose. Dilution of GLY with 1 or 1.25 M sucrose yielded better results compared with lower sucrose concentrations or the three-step GLY removal procedure.     

Effect of B-mercaptoethanol on the viability of IVM/IVF/IVC bovine embryos during long-distance transportation in plastic straws

Experiments were conducted to assess the effect of beta-mercaptoethanol (beta-ME) on the quality and viability of bovine blastocysts derived from in-vitro culture (IVC) of in-vitro matured and fertilized (TVM-IVF) oocytes during their transport between 2 distant places. Follicular oocytes were collected from ovaries obtained at a slaughterhouse and were cultured for 20 to 21 h in modified TCM-199. The IVM oocytes were fertilized in vitro with frozen-thawed spermatozoa. Fertilized oocytes were cultured for 7 d, and embryos that developed to the blastocyst stage were used for the experiments. The blastocysts, packed in straws with transportation medium that consisted of modified TCM-199 with HEPES equilibrated in air and supplemented with 20 % calf serum and 0, 10, 50, 100 or 150 microM beta-ME, were transported at 37 degrees C from Tokyo to Sapporo by air (18.3 h). The quality of blastocysts was assessed and ranked as excellent (A), good (B), fair (C) or poor (D) after transportation. The percentages of blastocysts ranked as A or B were significantly higher (P < 0.05) when the embryos were transported in beta-ME supplemented medium (80 to 100%) than when transported without beta-ME (54 %). Blastocysts ranked as A or B after transportation in medium with or without 150 microM beta-ME were nonsurgically transferred to synchronous recipients; 60 d after embryo transfer, 21/36 and 19/35 cows, respectively, were diagnosed as pregnant by palpation per rectum. These results indicate that beta-ME maintains the quality of bovine blastocysts in plastic straws for several hours without control of CO2 and that the concentration of beta-ME used in this experiment is not detrimental to the blastocysts.     

A new procedure for the cryopreservation of equine embryos

Early equine blastocysts and blastocysts were collected nonsurgically at six days post-ovulation. Thirty-two embryos were randomly assigned to a 2x2 factorial design. Factors were: 1) 0.5-ml straws or 1-ml glass ampules; and 2) plunging into liquid nitrogen (IN(2)) at -33 C or -38 C. Cryoprotectant, 10% glycerol in PBS plus 5% fetal calf serum (FCS) was added in two steps, 5% then 10%. Embryos were cooled at 4 C/min to -6 C and then seeded, 0.3 C/min to -30 or -35 C and 0.1 C/min to -33 or -38 C. Samples were thawed in 37 C water and glycerol removed in six steps, 10 min per step. Embryo quality and stage of development were evaluated prior to freezing, immediately post-thaw and after 24 h culture in Ham's F10 with 5% FCS. The mean post-thaw quality of embryos plunged at -33 C was superior (P<0.05) to that of embryos plunged at -38 C (2.0 vs 2.9). Embryos frozen in ampules and plunged at -38 C were of poorer quality (P<0.05) than those frozen in ampules and plunged at -33 C or frozen in straws and plunged at -33 C. After 24 h of culture, more embryos developed if frozen in straws compared to ampules, and plunging at -33 C resulted in higher quality embryos than plunging at -38 C. In Experiment 2, 23 embryos were packaged in straws and plunged at -33 C as described in Experiment 1. Six of the 23 surgically transferred frozen embryos were degenerate at thawing and the remaining 17 surgically transferred were via flank incision. Pregnancy rate at 50 days post-ovulation was 53% (nine of 17). Early blastocysts resulted in a higher (P<0.05) pregnancy rate (8 10 , 80%) than expanded blastocysts (1 7 , 14%).     

Effect of retinoids and growth factor on in vitro bovine embryos produced under chemically defined conditions

Experiments were conducted to investigate the beneficial effects of adding retinol (RT) and retinoic acid (RA) to bovine oocyte maturation media and insulin-like growth factor-I (IGF-I) to embryo culture under chemically-defined conditions. In Experiment 1.1, in vitro maturation (IVM) was performed in basic maturation media (bMM) and supplemented with 0.3microM RT or 0.5microM RA. For embryo development presumptive zygotes and embryos were placed in droplets of potassium simplex optimized medium (KSOM). Addition of RT and RA to bMM improved (p<0.05) blastocyst formation as compared with control treatments. In Experiment 1.2, using embryos originating from oocytes previously treated with RT and RA, the presumptive zygotes were placed in droplets of KSOM and embryos (2-4 cells) in droplets of fresh KSOM supplemented or not with IGF-I. The number of 2-4-cell stage embryos developing to the blastocyst and expanded blastocyst stages were greater (p<0.05) when embryo culture media was supplemented with IGF-I. In Experiment 2.1, IVM was conducted with bMM+FSH containing 0.3microM RT or 0.5microM RA. For embryo development, presumptive zygotes were placed in droplets of KSOM. Addition of RT or RA to IVM medium also enhanced (p<0.05) blastocyst formation. The supplementation of embryo culture media with IGF-I resulted in a greater number (p<0.05) of 2-4-cell stage embryos developing into blastocysts, expanded blastocysts and hatched blastocysts. In Experiment 2.2, using embryos originating from oocytes previously treated with RT and RA, presumptive zygotes were also placed in droplets of KSOM and embryos (2-4 cells) in droplets of fresh KSOM supplemented or not with IGF-I. The supplementation of embryo culture media with IGF-I resulted in a greater (p<0.05) number of 2-4-cell stage embryos developing to the blastocyst, expanded blastocyst and hatched blastocyst stages.     

Delipidating in vitro-produced bovine zygotes: effect on further development and consequences for freezability

To study the effect of partial removal of intracytoplasmatic lipids from bovine zygotes on their in vitro and in vivo survival, presumptive zygotes were delipidated by micromanipulation and cocultured with Vero cells in B2+10% FCS. Blastocyst rates of delipidated (n=960), sham (centrifuged but not delipidated, n=830) and control embryos (n=950) were 42.1, 42.3 and 39.9% respectively (P > 0.05). Day 7 blastocysts derived from delipidated zygotes had a mean of 123.9 +/-45.6 nuclei compared to 137.5+/-32.9 for control blastocysts (P > 0.05). The full-term development of delipidated blastocysts after single transfer to recipients was similar to that of control IVF blastocysts (41.2% vs 45.4% respectively). To assess the effect of delipidation on the embryo tolerance to freezing/thawing, delipidated (n=73), control (n=67) and sham (n=50) Day 7 blastocysts were frozen in 1.36 M glycerol + 0.25 M sucrose in PBS. After thawing, embryos were cocultured for 72 h with Vero cells in B2+10% FCS. Survival rates at 24 h were not significantly different between groups. However, in the delipidated group, the survival rate after 48 h in culture was significantly higher than in the control group (56.2 vs 39.8, P < 0.02), resulting in a higher hatching rate after 3 days in culture (45.2 vs 22.4, P < 0.02). Pregnancy rates for delipidated and control frozen/thawed embryos were respectively 10.5 and 22.2% (P > 0.05). Electron microscopic observations showed much fewer lipid droplets (and smaller) in delipated blastocysts than in controls. Taken together, our data show that delipidation of one cell stage bovine embryos is compatible with their normal development to term and has a beneficial effect on their tolerance to freezing and thawing at the blastocyst stage. This procedure, however, alters the developmental potential of such blastocysts, suggesting that maternally inherited lipid stores interfere with metabolic recovery after thawing.     

Open-pulled straw vitrification differentiates cryotolerance of in vitro cultured rabbit embryos at the eight-cell stage

The objective was to determine cryotolerance of in vitro cultured rabbit embryos to the open-pulled straw (OPS) method. Overall, 844 rabbit embryos at pronuclear, 2- to 4-cell, 8-cell, and morula/blastocyst stages were vitrified, and ≥ 1 mo later, were sequentially warmed, rehydrated, and subjected to continuous culture (n = 691) or embryo transfer (ET, n = 153). Embryos vitrified at the 8-cell stage or beyond had greater survival, expanded blastocyst and hatched blastocyst rates in vitro, and better term development than those vitrified at earlier stages. The 8-cell group had 70.1% expanded blastocysts, 63.7% hatched blastocysts, and 25.7% term development, as compared to 1.5-17.7%, 1.5-4.3% and 2.8-3.7% in the pronuclear, 2-cell and 4-cell embryos, respectively (P < 0.05). The expanded and hatched blastocyst rates in vitrified morula/blastocyst post-warming were higher than that in the 8-cell group; however, their term development after ET was similar (8-cell vs morula/blastocyst: 25.7 vs 19.4%, P > 0.05). Development after ET was comparable between vitrified-warmed embryos and fresh controls at 8-cell and morula/blastocyst stages (19.4-25.7 vs 13.7-26.6%, P > 0.05). For embryos at pronuclear or 2- to 4-cell stages, however, term rates were lower in the vitrified-warmed (2.8-3.7%) than in fresh controls (28.6-35.6%, P < 0.05). Therefore, cultured rabbit embryos at various developmental stages had differential crytolerance. Under the present experimental conditions, the 8-cell stage appeared to be the critical point for acquiring cryotolerance. We inferred that for this OPS cryopreservation protocol, rabbit embryos should be vitrified no earlier than the 8-cell stage, and stage-specific protocols may be needed to maximize embryo survival after vitrification and re-warming.     

Factors affecting in vivo viability of DNA-injected bovine blastocysts produced in vitro

In vitro matured and fertilized bovine ova were microinjected with pBL1, which consisted of the bovine beta-casein gene promoter, human lactoferrin cDNA and SV40 polyadenylation signal. Of the 2931 zygotes injected, 2505 (85.5%) survived 1 h after DNA injection and were cultured in 50-microl drops of CR1aa medium containing 3 mg/ml BSA under mineral oil at 39 degrees C, 5% CO2 in air. Cleaved (2- to 8-cell) embryos were selected at approximately 48 h after DNA injection and then cultured further in 50-microl drops of CR1aa medium supplemented with 10% (v/v) FBS. Blastocysts were classified into 4 quality grades and 3 developmental stages by morphological criteria. Then all but poor quality blastocysts were nonsurgically transferred to the uterus of heifers 7 to 8 d after natural estrus. Following transfer, the recipients were observed for signs of estrus, and pregnancy was confirmed by palpation per rectum at approximately 60 d of gestation. Although 72.0% (1804/2505 ) of the DNA-injected zygotes reached 2- to 8-cell stages only 5.2% (131/2505) developed to blastocysts. A total of 75 DNA-injected, in vitro cultured blastocysts were transferred to 59 recipients. When 2 blastocysts were transferred to a single recipient, only the better quality embryo was counted. The overall pregnancy rate was 30.5% (18/59 ) and reflected 1) an apparent correlation between the quality of embryos and the pregnancy rate. However, the difference was not statistically significant. 2) expanded blastocysts had a higher pregnancy rate (50.0%, 11/22 ) than early (13.3%, 2 15 ) or mid (22.7%, 5/22 ) blastocysts with a significant difference between expanded and early blastocysts (P < 0.05). 3) the pregnancy rate of DNA-injected blastocysts was higher when they were transferred at Day 7 (34.5%, 10/29 ) or 8 (36.8%, 7/19 ) than at Day 6 (9.0%, 1/11 ). The results indicate that the developmental stage of DNA-injected bovine embryos may be one of contributing factors in improving the pregnancy rate after transfer, although the effects of the quality and culture period of the embryos may not be inconsequential.     

Mouse zygote development in culture medium without protein in the presence of ethylenediaminetetraacetic acid

Development of zygotes from a hybrid-inbred (B6D2F1) and two random-bred (CD1 and CF1) strains of mice were compared after culture in several modifications of a simple, chemically defined medium based on Earle's Balanced Salts Solution. When cultured without the addition of protein or the chelating agent, ethylenediaminetetraacetic acid (EDTA), none of the zygotes reached the blastocyst stage. The addition of EDTA or protein significantly improved embryo development to blastocysts (p less than 0.05). The degree of improvement was dependent upon the strain of the female (85% or 91% for B6D2F1, 56% or 45% for CD1, and 19% or 28% for CF1, respectively). The addition of protein to the media in the presence of EDTA did not further improve embryo development. In all supportive conditions, zygotes from B6D2F1 females developed to blastocysts better than those from CD1 or CF1 females; embryos of the latter strain exhibited the lowest rates of development in vitro. Glycine and alanine (20 microM) partially substituted for EDTA; the decreased hybrid-inbred embryo development to blastocysts (20% and 26%, respectively) obtained in the presence of the amino acids suggested, however, that the stimulatory effect of EDTA on embryo development was other than as a source of fixed nitrogen. The rates of development observed with an alternate chelating agent, citric acid (less than or equal to 20% vs. 83% blastocysts, p less than 0.01), although better than the unsupplemented medium, were significantly less effective than EDTA-supplemented medium (83% blastocysts, p less than 0.01). The results of this study suggest that the protective effect of proteins in culture medium may be more important than their nutritive role.(ABSTRACT TRUNCATED AT 250 WORDS)     

Freezability of porcine blastocysts at different peri-hatching stages

The freezability of porcine peri-hatching stage blastocysts was investigated by the cryopreservation of embryos at -196 degrees C with 1.5 M glycerol and by thawing, followed by in vitro culture. Of 66 expanded blastocysts frozen, 34 (51.5%) developed in vitro after thawing, while only 2 (6.7%, P<0.05) of 30 earlier stage blastocysts survived freezing. After freezing of 85 hatched blastocysts with an embryonic diameter of 150 to 300 mum, 59 (69.4%) surviving embryos were obtained, whereas none of the 78 advanced staged hatched blastocysts (>300 mum) survived the cryopreservation. High post-thaw survival (32 39 , 82..1%) was obtained with in vitro-hatched blastocysts precultured in Whittingham's M-16 medium containing 12mg/ml bovine serum albumin (BSA). In contrast, none of the 14 in vitro-hatched blastocysts precultured in the M-16 medium supplemented with 15% fetal calf serum (FCS) survived freezing. Similarly 51 of 56 hatced blastocysts (diameter = 150 to 300 mum) precultured in the M-16 medium supplemented with BSA survived cryopreservation, compared with 3 of 26 embryos precultured in the medium supplemented with FCS (P<0.001). Because both groups of the embryos precultured with BSA or FCS possessed normal ability to develop after transfer (developmental rate = 61.1 and 93.3%), the improved freezability of the embryos precultured with BSA may relate to a favorable change of embryonic cell membranes during the culture period. It was concluded that in vitro-hatched blastocysts precultured in medium containing BSA and in vivo-hatched blastocysts at the appropriate stage of development could both tolerate deep freezing to -196 degrees C; however, a differece in the freezability of embryos between breeds of pig was suggested from a further experiment performed with German Landrace embryos.     

A comparison of Menezo's B2 and tissue culture Medium-199 for in vitro production of bovine blastocysts

The objectives of this study were, first, to evaluate the effectiveness of 2 culture media, Menezo's B2 (B2) and Tissue Culture Medium-199 (M-199), for the production of bovine blastocysts in a commercial embryo transfer program; and, second, to characterize the stage of development, quality grade and cell number of blastocysts produced in each medium. One-cell bovine embryos were produced using in vitro maturation and fertilization procedures. After fertilization, the embryos were co-cultured on Buffalo rat liver (BRL) cell monolayers in either B2 or M-199+1% BSA (M-199) medium. Both media were supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin. Embryo cultures were continued undisturbed to either Day 7 or Day 8 post-insemination. In the Day 7 cultures, all blastocysts were removed for evaluation on Day 7, and the remaining embryos were cultured for a further 24 h. Any additional blastocysts that formed were removed for evaluation and designated as Day 8 disturbed embryos. All blastocysts were classified for stage and quality grade. Embryos were fixed and stained for determination of cell number. Overall, the proportion of blastocysts was greater (P = 0.0003) with B2 medium (46%) than with M-199 (33%). This was due to a larger (P = 0.0001) proportion of blastocysts produced in B2 medium when cultures were left undisturbed for 8 d (50 vs 28% for B2 vs M-199). The proportion of blastocysts on Day 7 of culture tended to differ (P = 0.073) between media (33 vs 24% for B2 vs M-199). In addition, there were more (P = 0.007) blastocysts at advanced stages of development in B2 medium on Day 7. There was no effect of type of medium on the distribution of embryo quality grades on any day examined. The number of cells per blastocyst did not differ between media but did vary significantly (P < .05) with both stage and grade. In conclusion, B2 medium was superior to M-199 medium when used in a co-culture system with BRL cells for the production of bovine blastocysts.     

Improvement of transgenic cloning efficiencies by culturing recipient oocytes and donor cells with antioxidant vitamins in cattle

The present study was conducted to investigate effects of antioxidants during maturation culture of recipient oocytes and/or culture of gene-transfected donor cells on the meiotic competence of recipient oocytes, and the developmental competence and quality of the reconstructed embryos after nuclear transfer (NT) in cattle. Gene-transfected donor cells had negative effects on the proportions of blastocyst formation, total cell numbers, and DNA fragmentation indices of reconstructed embryos. Supplementation of either vitamin E (alpha-tocopherol: 100 microM) or vitamin C (ascorbic acid: 100 microM) during maturation culture significantly enhanced the cytoplasmic maturation of oocytes and subsequent development of embryos reconstructed with the oocytes and gene-transfected donor cells, but did not have synergistic effects. The supplementation of vitamin E during maturation culture of recipient oocytes increased the proportions of fusion and blastocyst formation of gene-transfected NT embryos, in which the proportions were similar to those of nontransfected NT embryos. When the gene-transfected donor cells that had been cultured with 0, 50, or 100 microM of vitamin E were transferred into recipient oocytes matured with vitamin E (100 microM), 50 microM of vitamin E increased the proportion of blastocyst formation and reduced the index of DNA fragmentation of blastocysts. In conclusion, gene-transfected donor cells have negatively influenced the NT outcome. Supplementation of vitamin E during both recipient oocyte maturation and donor cell culture enhanced the blastocyst formation and efficiently blocked DNA damage in transgenic NT embryos.     

Effects of quality and developmental stage on the survival of IVF-derived bovine blastocysts cultured in vitro after freezing and thawing

Factors affecting viability of IVF-derived bovine blastocysts after freezing and thawing were investigated. A total of 1,101 ova matured and fertilized in vitro were cultured under 2 different conditions, 1) in TCM-199 on granulosa cell monolayers at 5% CO(2) in air and 2) in synthetic oviduct fluid (SOF) medium without somatic cell support at 5% CO(2), 5% O(2), 90% N(2). All blastocysts that developed from the 2 different culture systems were individually classified into 4 grades of embryo quality and were then frozen by conventional slow freezing. Developmental rates of the IVF-derived ova to blastocysts and the survival rates of the frozen-thawed blastocysts were not different between the SOF medium (16 and 49%) and the co-culture system (13 and 61%, respectively). Survival of frozen-thawed blastocysts was affected by embryo quality in both the SOF and co-culture systems (P<0.001). Blastocysts produced in vitro were also individually classified into 3 developmental stages and were then cultured for 3 d in the co-culture system with granulosa cells after freezing and thawing. There was a difference in the survival rate of frozen-thawed embryos between blastocyst developmental stages (early vs mid, P<0.05; mid vs expanded, P<0.01; early vs expanded, P<0.001). The post-thawing survival rate of blastocysts frozen at Day 7 (62%) of culture was higher compared with that of Day 8 (45%), but there was no difference in survival rate between Day 7 and 8 of culture. The results indicate that the quality and developmental stage of blastocysts are important factors influencing their survival after freezing and thawing.     

Effect of sericin on preimplantation development of bovine embryos cultured individually

The silk protein sericin has been identified as a potent antioxidant in mammalian cells. This study was conducted to examine the effects of sericin on preimplantation development and quality of bovine embryos cultured individually. When two-cell-stage embryos were cultured individually for 7 days in CR1aa medium supplemented with 0, 0.1, 0.5, or 1% sericin, rates of total blastocyst formation and development to expanded blastocysts from embryos cultured with 0.5% sericin were higher (P < 0.05) than those from embryos cultured with 0 or 1% sericin. When embryos were cultured individually for 7 days in the CR1aa medium supplemented with 0 or 0.5% sericin under two oxidative stress conditions (50 or 100 μm H₂O₂), the addition of sericin significantly improved the blastocyst formation rate of embryos exposed to 100 μm H₂O₂. However, the protective effect of sericin was not observed in development of embryos exposed to 50 μm H₂O₂. When embryos were exposed to 100 μm H₂O₂ during culture, the DNA fragmentation index of total blastocysts from embryos cultured with 0.5% sericin was lower than blastocysts derived from embryos cultured without sericin (4.4 vs. 6.8%; P < 0.01). In conclusion, the addition of 0.5% sericin to in vitro culture medium improved preimplantation development and quality of bovine embryos cultured individually by preventing oxidative stress.     

Developmental competence of porcine parthenogenetic embryos relative to embryonic chromosomal abnormalities

Parthenogenetically activated (PA) embryos exhibit delayed development, a lower blastocyst rate, and less successful development in vitro compared to in vitro fertilized (IVF) embryos. To investigate the possible mechanisms for unsuccessful parthenogenetic development, this study analyzed the chromosome abnormalities and developmental potential of porcine PA embryos. Mature oocytes were electrically activated and cultured in Porcine Zygote Medium-3 (PZM3) supplemented with 3 mg/ml BSA for 6, 7, or 8 days. The percentage of PA blastocysts was lower than that of IVF embryos on days 6 and 7 (16.4 +/- 7.4 vs. 28.7 +/- 3.7; 10.9 +/- 2.8 vs. 21.5 +/- 4.7, P < 0.05; respectively), and the PA blastocysts had significantly fewer nuclei than IVF blastocysts (23.2 +/- 1.8 vs. 29.7 +/- 0.8; 29.7 +/- 3.3 vs. 32.0 +/- 2.4, P < 0.05). The percentage of abnormal PA embryos (including embryos with condensed nuclei, arrested embryos and fragmented embryos) was higher than that of IVF embryos (PA: 52.9 +/- 12.8 vs. 16.4 +/- 7.4 on day 6), and increased with culture time (71.9 +/- 12.1 vs. 10.9 +/- 2.8. on day 7,and 75.0 +/- 22.6 vs. 12.1 +/- 2.3 on day 8, P < 0.05). The Day-6 PA blastocysts (n = 147) were divided into three classes according to the total number of nuclei (<20, 20-39, >40) and into three groups according to the morphological diameter (<150, 150-180, >180 microm). Of the haploid blastocysts, 56.1% had less than 20 nuclei, and 71.5% were less than 150 microm in diameter. Of all (114) blastocysts suitable for analysis, 55.5% displayed chromosomal abnormalities. Among chromosomal abnormalities in PA blastocysts, haploid blastocysts were most prevalent (43.6%), while polyploidy (4.4%) and mixoploidy (7.7%) embryos were less prevalent. Chromosomal abnormalities of porcine PA embryos might contribute to a higher rate of abnormal embryonic development. We suggest that a careful consideration should be given when using the blastocysts with smaller size, and establishing the optimum culture condition for PA embryos development in vitro.     

Effects of varying the holding temperature and interval from collection to freezing on post-thaw development of bovine embryos in vitro

The objective of this study was to evaluate the in vitro development of frozen-thawed bovine embryos held at room temperature or refrigerated for 2, 6 or 12 h prior to freezing. After recovery, embryos were randomly assigned to be placed in holding media for 2 h (n=131), 6 h (n=136) or 12h (n=133) prior to freezing. Approximately one-half of the embryos were refrigerated (5 degrees C; n=203) while the remaining half were held at room temperature (22 degrees C; n = 197) until freezing. Embryos were frozen in 10% ethylene glycol and stored in liquid nitrogen. After thawing, embryos were cultured for 72 h in Ham's F-10 media supplemented with 4% fetal bovine serum. Embryos were evaluated for quality and stage of development prior to freezing and after culture. At the end of culture, it was determined if each embryo had developed beyond the stage recorded prior to freezing and if the embryo had hatched from the zona pellucida. The percentage of embryos that developed during culture was greater (P < 0.001) for Grade 1 (81%) than for either Grade 2 (65%) or Grade 3 (48%) embryos. Likewise, a greater proportion (P < 0.001) of Grade 1 embryos developed to hatched blastocysts (60%) than either Grade 2 (40%) or Grade 3 (24%) embryos. The holding temperature from collection to freezing did not influence embryo development, regardless of the interval from embryo collection to freezing. The proportion of embryos that developed to expanded blastocysts and hatched was greater (P < 0.005) for embryos held 2 h prior to freezing (64%) than for embryos held for 12 h (33%). Hatching rate of embryos held 6 h prior to freezing (54%) tended (P < 0.08) to be lower than the hatching percentage for embryos held for 2 h. Thus, post-thaw embryonic development was impaired the longer embryos were held prior to freezing and temperature during the interval from collection to freezing did not affect post-thaw development.     

Effects of fetal calf serum, phenazine ethosulfate and either glucose or fructose during in vitro culture of bovine embryos on embryonic development after cryopreservation

This study investigated effects of hexoses, fetal calf serum (FCS), and phenazine ethosulfate (PES) during the culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. The basal, control medium was chemically defined (CDM) plus 0.5% fatty acid-free BSA. In vitro-produced bovine zygotes were cultured in CDM-1 with 0.5 mM glucose; after 60 hr, 8-cell embryos were cultured 4.5 days in CDM-2. The 8-cell embryos were randomly allocated to a 2 x 3 x 2 x 3 factorial experimental design with two energy substrates (2 mM glucose or fructose); three additives (0.3 microM PES, 10% FCS, and control); two cryopreservation methods using no animal products (conventional slow freezing or vitrification); and semen from three bulls with two replicates for each bull. A total of 1,107 blastocysts were produced. Fructose resulted in 13% more blastocysts per oocyte than glucose (37.2% vs. 32.9%), and per 8-cell embryo (51.3% vs. 45.3%; P < 0.01). No differences were found for additives (P > 0.1) control, FCS, or PES for blastocysts per oocyte or per 8-cell embryo. There was a significant interaction (P < 0.05) between additives and hexoses for blastocyst production; although trends were similar, the benefit of fructose compared to glucose was greater for controls than for FCS or PES. Culture of embryos with PES, which reduces cytoplasmic lipid content, improved cryotolerance of bovine embryos; post-cryopreservation survival of blastocysts averaged over vitrification and slow freezing (between which there was no difference) was 91.9%, 84.9%, and 60.2% of unfrozen controls (P < 0.01) for PES, control, and FCS groups, respectively.     

小鼠2—细胞胚胎单个卵裂球体外培养及其发育形成囊胚的玻璃化冷冻保存技术的研究（简报）

The present experiments were designed to study the effects of glucose, EDTA, glutamine on the in vitro development of single blastomeres from 2-cell embryos in mouse, and the efficiency of cryopreservation of blastocysts from single blastomers with different vitrification. Single blastomeres derived from female ICR x male BDF1 2-cell embryos were cultured in mKRB with or without glucose, EDTA and glutamine, respectively. The expanded blastocyst rates were significantly different between in mKRB with glucose and without glucose (34% vs 65%); The blastomeres were cultured in mKRB with EDTA and glutamine but glucose, the expanded blastocyst rate (90%) was significantly higher than other groups. The blastocysts derived from single blastomeres were vitrified in liquid nitrogen after equilibration in GFS40 for 0.5-2 min, the survival rate 24%-51%. The blastocysts were pretreated in mPBS with 10% glycerol for 5 min, followed by exposure to GFS40 at 25 degrees C for 0.5 min, then vitrified in liquid nitrogen(two-step method), the survival rate was 61%. However, the survival rates increased to 64% and 70% when the blastocysts were vitrified(one-step method) ater equilibration in EFS40 at 25 degrees C for 0.5-1 min.     

Vitrification of porcine embryos at various developmental stages using different ultra-rapid cooling procedures

In this study, three different vitrification systems (open pulled straw: OPS; superfine open pulled straw: SOPS; and Vit-Master technology using SOPS: Vit-Master-SOPS) were compared in order to investigate the influence of cooling rate on in vitro development of vitrified/warmed porcine morulae, early blastocysts, or expanded blastocysts. Embryos were obtained surgically on Day 6 of the estrous cycle (D0 = onset of estrus) from weaned crossbred sows, classified and pooled according their developmental stage. A subset of embryos from each developmental stage was cultured to evaluate the in vitro development of fresh embryos; the remaining embryos were randomly allocated to each vitrification system. After vitrification and warming, embryos were cultured in vitro for 96 h in TCM199 with 10% fetal calf serum at 39 degrees C, in 5% CO(2) in humidified air. During the culture period, embryos were morphologically evaluated for their developmental progression. The developmental stage of embryos at collection affected the survival and hatching rates of vitrified/warmed embryos (P < 0.001). The vitrification system or the interaction of vitrification system and developmental stage had no effect on these parameters (P > 0.05). Vitrified expanded blastocysts showed the best development in vitro (P < 0.001), with survival and hatching rates similar to those of fresh expanded blastocysts. The hatching rate of fresh morula or early blastocyst stage embryos was higher than their vitrified counterparts. In conclusion, under our experimental conditions, cooling rates greater than 20,000 degrees C/min, as occurs when SOPS or Vit-Master-SOPS systems are used, do not enhance the efficiency of in vitro development of vitrified porcine embryos.